With a conventional light microscope three-dimensional structures typically cannot be successfully imaged. Ordinarily, the resultant image consists of a sharp image of an in-focus region of a three-dimensional structure as well as defocused images of the structures above and below the in-focus region. A conventional light microscope is unable to reject out-of-focus detail.
Confocal microscopes have been developed which optically section a three-dimensional structure to provide in-focus images of individual layers or strata of the structure. These individual layers can be subsequently combined to form an in-focus three-dimensional image of the complete volume structure. Unfortunately, the light budget of confocal microscopes is generally poor when incoherent light sources are used. Laser scanning confocal microscopes can achieve a very shallow depth of focus, but typically require expensive apparatus and an illuminating/imaging pinhole through which the laser light is focused.
In U.S. Pat. No. 5,381,236 to Morgan, an optical sensor is described which is used to determine the range (distance) of individual features of a three-dimensional structure. The sensor has a periodic patterned light source that illuminates the structure and is reversible (i.e. the pattern is phase shifted 180°). An array of detector elements aligned to the pattern of the light source is used to detect an image of the pattern and the reversal of the pattern illuminating the structure. As the pattern is generally only imaged well on those parts of the structure which are themselves in focus, this enables the range (distance) of in-focus parts of the structure to be determined. A potential shortcoming of the apparatus and method described in Morgan can be that, in order to operate properly, the individual elements of the detector should be exactly aligned with and matched to the pattern of the light source. In practice this has been found to be almost impossible to achieve.
One solution to the problem is offered in U.S. Pat. No. 6,376,818 to Wilson et al., the disclosure of which is incorporated herein by reference in its entirety. The technique discussed in Wilson et al. involves the superposition of a periodic pattern of transparent and non-transparent stripes onto the object of interest. At least three images are recorded at different spatial phases of the pattern by means of a microscope with a shallow focal depth. A three-dimensional image composed of only in-focus detail is then derived from the recorded images (this technique is known in some circles as a “full-focus” technique). An exemplary system of this variety is the OPTIGRID™ microscopy system, available from Thales Optem, Fairport, N.Y.
Unfortunately, conventional microscopy imaging masks utilized, for example, by Wilson et al. can leave residual patterning in the final image. The degree of the residual pattern is a function of the intensity profile in the illumination pattern. Such residual patterning is undesirable since it raises doubt as to whether the pattern is part of the specimen or an artifact of the imaging process.
Because of the accessibility and easy fabrication, ronchi gratings (i.e., square waves) have been used in structured illumination. However, when imaged through band-limited systems (such as an optical system) the resultant image has residual patterns. This occurs because the ronchi grating passed through a linear band-limited system is the convolution of the ronchi grating and the point spread function (PSF) of the optics. The result is a square wave with rounded shoulders. It is at these shoulders where residual lines occur. For this reason, it is desirable with some systems to utilize “stripes” in the illumination pattern that vary sinusoidally in intensity.
One issue that can arise with full-focus techniques, and particularly those that utilize a set of sinusoidally-varying stripes in the pattern, is related to the calibration of the system. Generally speaking, microscopy errors can occur if the system is not precisely calibrated. In particular, it can be important to calibrate the location of a mask or similar device that produces the pattern of stripes, particularly as the mask is positioned for different phases. It would be desirable to improve performance of full-focus systems via new calibration techniques.